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Microbiome of smear ripened cheese and its changes in dependence on the stages of production
Jandová, Karolína ; Mikešová,, Martina (referee) ; Trachtová, Štěpánka (advisor)
The theoretical part describes soft smeared cheeses, then deals with the technology of manufacturing said cheese with importance on the development of microbiome and describes the most commonly wanted microorganisms and contaminants. Shortly also deals with legislation requirements on the microbial quality of these chesses. The experimental part studies the development of microbiome and contamination by fungi of soft smeared cheese. Firstly, samples were collected in manufacture from each step of manufacturing. In the first part, microbial and microscopical assessment was done. In the second part, DNA was isolated from samples by a commercial kit and phenol-chloroform extraction. The concentration and purity of isolates were determined by spectrophotometry. By qPCR of the DNA isolates the main microbiome and contamination by fungi were examined. As part of the microbial and microscopical assessment, the expected bacteria were determined, and several species of yeast were found, instead of the expected one. Mold contamination was also found. Using molecular diagnostic techniques, the presence of bacteria, yeasts, fungi and the genera Brevibacterium and Candida was proved.
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Testing of primers for real-time PCR-HRM analysis of fruit products containing one fruit species
Boháčová, Barbora ; Dzurendová, Simona (referee) ; Fialová, Lenka (advisor)
Determining the authenticity of fruit products, which are often counterfeited by substituting part of a more expensive fruit with a cheaper but botanically similar fruit, is a current topic in the food industry. A prime example is the dilution of apricot products with peach puree. This study focuses on testing specific primers AGS18 and PdCass to reveal the true proportion of apricot content in model products mixed with peach puree using PCR analysis followed by HRM analysis. Testing of these primers for authenticity determination revealed limited utility of AGS18 primers due to the formation of small amount or no specific products during reactions with fruit DNA. PdCass primers required PCR condition optimization, but subsequently, specific DNA sequences from fruit leaves could be amplified in sufficient quantities. PCR analysis of DNA from model products with PdCass primers provided specific products in samples with apricot content of 70% or less. HRM analysis of samples and calculation of GCP did not distinguish purees with 0%, 10%, and 30% apricot content. However, purees with 50% and 70% apricot content exhibited significantly different melting curves compared to other samples.
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Selective isolation of the genus Bifidobacterium bacteria from foods
Mizerovská, Lucie ; Šárka, Havlíková (referee) ; Rittich, Bohuslav (advisor)
Probiotic lactic acid bacteria (LAB) are very often used in food procesing industry, such as milk products, cheese and fermentsd salami production in nova days. In diploma thesis were tested symbiotic food supplements from different producers. Bacterial DNA was isolated from crude cell lysates of six food suplements by magnetic particles P(HEMA-co-GMA). PCR-ready DNAs were isolated. from all products The detection of Bifidobacterium bacteria identified by PCR was in agreement with those declared by the manufacturers. Magnetic particles with immobilized antibodies against Bifidobacterium were used in the next part of thesis. These particles were used for the isolation of target cells from two products with cell identification by genus specific PCR.
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Selective isolation of of the genus Lactobacillus bacteria from foods
Novotná, Eva ; Šárka, Havlíková (referee) ; Rittich, Bohuslav (advisor)
Probiotic lactic acid bacteria of genus Lactobacillus play an important role in the digestive tract of human. They are used in food processing and they are the part of food supplements. Lactic acid bacteria of the genus Lactobacillus can be identificated by polymerase chain reaction (PCR). Bacterial DNA was isolated from cell lysates of 4 synbiotic food suplements by magnetic particles P(HEMA-co-GMA). Isolated DNA was amplified by genus-specific and species-specific primers. Magnetic particles with immobilized antibodies against Lactobacillus bacteria were used in the next part of thesis. These particles were used for isolation target cells from products with their identification by genus specific PCR.
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Identification of bacteria of Lactobacillus acidophilus species in probiotic products
Sznapková, Veronika ; Trachtová, Štěpánka (referee) ; Španová, Alena (advisor)
Probiotic lactic acid bacteria (LAB) are an important part of fermented dairy products, pharmaceuticals and food supplements. At present, rapid and accurate identification of bacteria is carried out using molecular biological methods based on DNA amplification. The aim of the thesis was to identify by non-cultivation bacteria of genus Lactobacillus and bacteria of species Lactobacillus acidophilus in complex matrices at total of seven different food supplements. Total DNA was isolated from crude cell lysates using magnetic carrier P(HEMA-co-GMA). Amplificability of DNA was verified by PCR using primers specific for the domain Bacteria. In next step isolated DNA was amplified using primers specific for the genus Lactobacillus and species Lactobacillus acidophilus to demonstrate the presence of this bacterial genus and species declared by the producers. The results of bacteria identification obtained by PCR were compared with declared specification given by the producers.
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Probiotics and their use in food industry
Diado, Aleksandra ; Rittich, Bohuslav (referee) ; Španová, Alena (advisor)
Probiotic bacteria are defined as live microorganisms, which when consumed in the determining quantities, have healthy and beneficial effects. Most of probiotics belongs to the genera Lactobacillus and Bifidobacterium. These and other genera of microorganisms are successfully used in industry, including food industry at present. Probiotics are used primarily in dairy products and food additives in food idustry. Probiotic bacteria, like other organisms, can be to identifie by PCR method that allows amplifying specific regions of DNA. Polymerase chain reaction was performed after DNA isolation from bacterial cultures of three strains using phenol extraction method. PCR specific for the domain Bacteria and genus-specific PCR were used for the confirmation of the presence of bacteria of the genus Lactobacillus.
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PROBIOTIC GENES OF SIGNIFICANT LACTIC ACID BACTERIA IN FOOD
Konečná, Jana ; Ševčovičová,, Andrea (referee) ; Doškař, Jiří (referee) ; Španová, Alena (advisor)
Isolation of deoxyribonucleic acid (DNA) is an important step in the molecular diagnostics of microorganisms. A high quality of isolated DNA is necessary for DNA amplification by the polymerase chain reaction (PCR). The conventional DNA isolation using phenol chloroform extraction and DNA precipitation in ethanol is time consuming and requires the use of toxic phenol. Magnetic separation techniques using magnetic solid particles are one of modern methods to speed up the nucleic acids isolation. The aim of this work was to use two different types of magnetic particles for solidphase DNA extraction. The amounts of DNA in separation mixtures were measured using ultraviolet spectrophotometry (UV). The first experimental conditions were tested on chicken erythrocytes DNA. Phosphate buffer (pH 7, 7.6 and 8) was used for adsorption of DNA on magnetic particles. It was shown that approximately almost one half of DNA was adsorbed to the particles. The elution conditions of DNA were also optimized. Secondly, bacterial DNA was tested. This DNA eluted from the particles was in PCR ready quality. High resolution melting (HRM) curve analysis is a simple, low-cost method for amplicon discrimination and easy connection with real-time polymerase chain reaction (PCR). In this contribution, we report rapid species identification of strains belonging to the Lactobacillus group using HRM-PCR. Three different DNA isolation methods were used in this work: phenol extraction, separation using magnetic particles and commercial kit. Ten sets of targeted gene fragments primers (LAC1 – LAC2, LAC2 – LAC4, P1V1 – P2V1, Gro F – Gro R, 3BA-338f – Primer 1, V1F – V1R, CHAU - V3F – CHAU - V3R, CHAU - V6F – CHAU - V6R, poxcDNAFw – poxPromRVC, poxcDNAFw – poxPromRVT) were tested for amplification of the 16S rRNA gene. Use of GroF/R and LAC2/4 primers pairs successfully identify strains belong to the Lactobacillus group. The variance between used extraction methods for evidence of HRM curves was found.
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Sample preparation for DNA analysis from foods of plant origin
Silná, Renata ; Rittich, Bohuslav (referee) ; Kovařík, Aleš (advisor)
The isolation of high quality DNA is nessecary for many molecular biology applications. However, plant DNA contains high amonts of polysaccharides, polyphenols and various secondary metabolites, which decrease yield and quality of isolated DNA. The aim of this study was preparation of samples and different food matrices for DNA isolation DNA by magnetic particles. It was about 5 species of vegetable and 10 species of processed plant food. Homogenization of samples was performed in CTAB buffer. Isolation of plant DNA was performed by magnetic particles covered with carboxyl groups. All DNAs were isolated in conventional PCR qualities using primers for 700 bp amplicons, in the case of heat processed products for 220 bp ampilicons and for real time PCR. The efficiancy of separation of magnetic particles with DNA by magnetic separator and magnetic needle was compared. It was find out that DNA of higher purity was isolated using magnetic needle. The micromethod of isolation of plant DNA from homogenates with CTAB with magnetic particles is suitable for different processed food.
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The analysis of DNA isolated from different types of probiotic products using real-time PCR and HRM analysis
Sedláková, Lucie ; Rittich, Bohuslav (referee) ; Trachtová, Štěpánka (advisor)
The aim of this diploma thesis was to introduce real-time PCR with high-resolution melting analysis for Bifidobacterium species. Currently a small number of publication, dealing with identification of Bifidobacterium species using high-resolution melting analysis, is available. According to publications dealing with identification of lactic acid bacteria were selected primers P1V1 and P2V1, LAC1 and LAC2, LsppUPF and LsppUPR, V3F and V3R, V6F and V6R. Using this primers bacterial DNA was amplified by real-time PCR with high-resolution melting analysis. After evaluation of the measured results efficiency of selected primers was verified on DNA izolated from complex sample of probiotic product. After further optimisation real-time PCR with high-resolution melting analysis could be suitable using selected primers for Bifidobacterium species.
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